This project involves the study of a rapidly emerging group of immune receptors. Many families of inhibitory immune receptors have recently been identified in both mice and humans. Interestingly, within each of these inhibitory families of receptors, there are proteins that have lost the inhibitory domains. Instead these receptors have gained a positively charged acid within their transmembrane domain, suggesting that they may interact with signal transduction chains and transmit positive signals. In this project, we study the signal transduction and biochemistry of both the positive and negative regulators of immune cell function. Through the study of the positive receptors, we and others demonstrated the association of some of these receptors with the novel signal transduction chain DAP12. Since then we have been characterizing the biochemistry of the DAP12 signal transduction pathway. This work has included demonstration of the kinases involved in the early signaling of DAP12, delineation of the adaptors involved, and study of the regulation of these pathways. In addition, to DAP12, we are beginning the study of DAP10, a second chain known to associate with receptors within NK cells and monocytes that is located just 130 bp from DAP12 on Chromosome 19. DAP10 contains a tyrosine based motif unique from that of DAP12. This motif (Y*xNM) suggests interaction with both the phosphatidylinositol 3 kinase (PI3K) and the adaptor Grb2. We are now preparing to dissect the signaling of DAP10 in an effort to fully understand the role of these chains within NK cells, monocytes and dendritic cells. Our studies of paired receptor systems has now largely shifted to the study of the Triggering Receptors Expressed on Myeloid cells (TREM). TREM-1 has recently been shown to be involved in the amplification of signals leading to septic shock. TREM-2 has been reported to be involved in the maturation of dendritic cells. We recently described TREM Like Transcript-1, a putative inhibitory receptor within the TREM cluster. TLT-1 transcripts have been found in various myeloid cells by RT-PCR, however, northern blotting demonstrates abundant TLT-1 expression only in platelets and bone marrow. Immunohistochemical and immunofluorescence studies show that bone marrow TLT-1 is derived exclusively from megakaryocytes. This finding makes TLT-1 only the second inhibitory receptor described on peripheral blood platelets (the other being PECAM-1), and the only inhibitory receptor expressed exclusively in platelets and megakaryocytes. The subcellular localization and regulation of TLT-1 in megakaryocytes and platelets suggests that TLT-1 is prepackaged into the platelet alpha granules by megakaryocytes for rapid surface expression after platelet degranulation. These findings suggest TLT-1 may be involved in the production and release of alpha granules and/or the